Wnt proteins are secreted signaling molecules that regulate many fundamental aspects of cellular functions in embryogenesis and tissue homeostasis. Deregulated Wnt signaling has been implicated in many human diseases such as cancer, osteoporosis and degenerative disorders. The study of the mechanisms of Wnt signaling has critical importance for understanding basic biology and human health. Wnt signaling is initiated by two types of transmembrane receptors that constitute the Wnt receptor complex. One is a member of the Frizzled (Fz) family of serpentine receptors, while the other is a single transmembrane receptor belonging to the LDL receptor related protein (LRP) family, LRP5 or LRP6. In addition to Wnt proteins, many distinct extracellular ligands appear to engage the Fz and/or LRP5/6 receptors and activate or inhibit Wnt signaling during embryonic development and under pathological conditions. The relationships between these Wnt agonists/antagonists with Fz and LRP5/6 receptors remain poorly understood, and are the focus of this application. Three related aims for formulated. Aim1 is to study R-spondin proteins, a novel family of agonists that activate Wnt signaling. R-spondin proteins have roles in vertebrate embryogenesis and are mutated in hereditary diseases. R-spondin proteins are also being developed as cancer therapeutic agents. But the relationships among R-spondin, Wnt and Fz-LRP5/6 receptors are debated and unresolved. Experiments are proposed to address these issues and to identify additional Rspo-binding factors/receptors. Aim 2 is to study the mechanism and function of a novel transmembrane protein, M15, that inhibits Wnt/2-catenin signaling. We have performed a functional cDNA expression screen in Xenopus embryos for genes that promote anterior formation. We identified a novel transmembrane protein, termed M15, that functions as a potent Wnt antagonist. M15 exhibits specific expression in the Spemann's organizer and also during somitogenesis. We propose experiments to study the molecular mechanism of M15 in inhibition of Wnt signaling and the biological function of in head/anterior development and somite formation. Aim 3 is to study M15L function and to identify additional novel cDNAs involved in anterior-posterior patterning. M15- like (M15L) is the other member of the M15 family we identified in Xenopus and human. It is a Wnt antagonist and has head-inducing activity in Xenopus embryos, but exhibits noticeable difference in Wnt inhibition specificity compared to M15. We plan to study the expression and function of M15L and uncover similar and/or distinct roles of M15 and M15L during Xenopus development. Additionally, based on our success with the isolation of M15, we plan to continue a more comprehensive functional cDNA expression screen for other novel genes that are involved in anterior-posterior patterning. These molecular and embryological experiments together will likely advance our understanding of the regulation of the Wnt receptor complex by novel agonists and antagonists in vertebrate development and human diseases. PUBLIC HEALTH RELEVANCE: Cell growth and differentiation are regulated by the interaction between extracellular signaling molecules and their receptors on the cell surface. Defects in this interaction often cause human cancers and diseases. This proposal aims to understand the molecular nature of the interaction between a key family of signaling molecules and their receptors that have been associated with many diseases.